PARENT SESSION

(T728) EFFECT OF AMH, BMP-15 AND GDF-9 ON FSH-INDUCED DIFFERENTIATION OF SHEEP GRANULOSA CELLS.

Campbell, Bruce¹, Sharma, Shweta², Shimasaki , Shunichi², Baird, David³, ¹ University of Nottingham, Nottingham, UK² University of California, San Diego, C.A.³ University of Edinburgh, Edinburgh, Scotland, UK

ABSTRACT- In sheep, mutations in growth differentation factor-9 (GDF-9: FecG) and bone morphogenic protein-15 (BMP-15: FecX) result in infertility in homozygotes and increases in ovulation rate in heterozygotes. Similarly, the FecB mutation to the BMP-1B receptor (BMPR-1B) results in increases in ovulation rate. The aim of this study was to examine the effect of GDF-9 and BMP-15, and another potential ligand of the BMPR-1B receptor, anti-mullerian hormone (AMH), on FSH-induced differentiation of sheep granulosa cells (GC) . Serum-free cultures of GC isolated from small (1-3 mm) antral follicles were established under conditions known to induce cellular differentiation (Campbell 1996 JRF 106) with a range of doses of rhGDF-9, rhBMP-15 (0.05-250 ng/ml) and rhAMH (0.005-50 ng/ml) for 8 days. rhBMP-6 was also included as a positive control. All cells were cultured with FSH (1 ng/ml) and responses were tested at two doses of IGF-1 (1 & 10 ng/ml). Media was changed every 48 h and assayed for estradiol (E2) and progesterone (P4). Viable cell number was determined on Day 8. As expected, BMP-6 stimulated E2 and inhibited P4 production in a dose responsive manner (P<0.01). In contrast, GDF-9, BMP-15 and AMH had dose responsive effects on E2 production without effecting P4. The E2 response to GDF-9 and BMP-15 was biphasic with higher doses (50 & 250 ng/ml) being inhibitory and lower doses (0.5 ng/ml) being stimulatory (P<0.01). AMH inhibited E2 production in a dose responsive fashion (P<0.01). IGF had a significant effect on steroid production (P<0.001) but there was no interaction between dose of IGF and dose of GDF-9, BMP-15 or AMH. These factors had no effect on cell number. We conclude that these recombinant human preparations are bioactive in sheep and that by selectively modulating the sensitivity of the granulosa cells to FSH, these local factors may provide mechanisms to explain the alterations in ovulation rates observed in sheep with Fec mutations.

KEY WORDS: granulosa cell, BMP-15, AMH, GDF-9